SUMMARY Phosphatidate (PA) phosphatase (PAP) is an evolutionarily conserved enzyme that plays a key role in lipid homeostasis by controlling the cellular levels of its substrate, PA, and its product, diacylglycerol. These lipids are essential intermediates for the synthesis of triacylglycerol and membrane phospholipids; they also function in phospholipid synthesis gene expression, lipid droplet formation, and vesicular trafficking. The importance of PAP to lipid homeostasis and cell physiology is exemplified in yeast, mouse, and human by a host of cellular defects and lipid-based diseases associated with loss or overexpression of enzyme function. In yeast, loss of Pah1 PAP results in a massive expansion of the nuclear/ER membrane; this is ascribed to increases in PA content and phospholipid synthesis that occur at the expense of triacylglycerol synthesis. The increase in phospholipid synthesis is associated with the derepression of phospholipid synthesis gene expression, whereas the reduction in the synthesis of triacylglycerol is associated with a decrease in lipid droplet formation. Lipin PAP deficiency in mouse and human causes rhabdomyolysis, and deficiency in the mouse is also characterized by hepatic steatosis during the neonatal period, lipodystrophy, insulin resistance and peripheral neuropathy. The overexpression of lipin 1 PAP in mouse results in increased lipogenesis and obesity. Human lipin 2 PAP deficiency causes chronic recurrent multifocal osteomyelitis and congenital dyserythropoietic anemia, whereas genetic variations in the human LPIN2 PAP gene are associated with type 2 diabetes. PAP is a peripheral membrane protein that must translocate from the cytosol to the nuclear/ER membrane in order to convert PA to diacylglycerol. This conserved process is governed by phosphorylation/dephosphorylation of the enzyme. In the cytosol, PAP is phosphorylated by multiple protein kinases that causes its retention in this cellular compartment. The membrane association of PAP requires is dephosphorylation by a conserved protein phosphatase complex (e.g., Nem1-Spo7 in yeast, CTDNEP1-NEP1-R1 in mouse and human). Besides its location, the phosphorylation of PAP inhibits its activity but stabilizes it to proteasomal degradation; dephosphorylation has the opposite effects. The work proposed in this MIRA application, which builds on our prior work made possible by the advantages of the yeast model, will gain understanding into the structure- function, mode of action and phosphorylation/dephosphorylation-mediated regulations of Pah1 PAP and the Nem1-Spo7 protein phosphatase complex. We will pursue rigorous experimental approaches that combine biochemistry and molecular genetics to shed light on how the proportional synthesis of triacylglycerol and membrane phospholipids is controlled. Based on the conserved nature of the Nem1-Spo7/Pah1 phosphatase cascade, the information gained from our studies with yeast is expected to be relevant in human.